The mechanism and the relationship between structure and mechanism in three pyridine nucleotide-disulfide oxidoreductases, lipoamide dehydrogenase, glutathione reductase, and thioredoxin reductase are being studied. Electron transfer catalyzed by each of these enzymes involves the FAD prosthetic group and an oxidation-reduction active disulfide of a cystine residue. The active site cystine in the three flavoproteins conforms a tight loop in the polypeptide chain. There is a high degree of amino acid sequence homology between lipoamide dehydrogenase and glutathione reductase and between pig heart (eukaryote) and Escherichia coli (prokaryote) lipoamide dehydrogenase. Two-electron reduction of pig heart lipoamide dehydrogenase generates 2 thiols, only one of which reacts with iodoacetamide rapidly. During the reaction charge transfer is abolished. The reactive cysteine residue is the one nearer the amino terminus. The monoalkylated derivative is catalytically inactive toward reduced or oxidized lipoamide but is some 2-fold activated as a transhydrogenase. The derivative binds NAD tightly at pH 7.6 and the absorbance at 450 nm is bleached. Spectral analysis indicates that the flavin has formed an adduct at the 4a position of the isoalloxazine ring. There is a base at the active site of pig heart lipoamide dehydrogenase which is protonated upon reduction. This protonation serves to activate the dihydrolipoamide for nucleophilic attack on the active center disulfide. As such a mechanism predicts, the rate of reduction of the enzyme by dihydrolipoamide (extrapolated to infinite concentration) is independent of pH over the range 5.5 to 7.6. BIBLIOGRAPHIC REFERENCES: "Differential Reactivity of the Two Active Site Cysteine Residues Generated on Reduction of Pig Heart Lipoamide Dehydrogenase", C. Thorpe and C.H. Williams, Jr. (1976) J. Biol. Chem. 251, 3553-3557. "Measurement of the Oxidation-Reduction Potentials for Two Electron and Four Electron Reduction of Lipoamide Dehydrogenase from Pig Heart", R.G. Matthews and C.H. Williams, Jr. (1976) J. Biol. Chem. 251, 3956-3964.